Chicken Myelomonocytic Growth Factor Research Articles

Hematopoietic growth factor glycosylation. Multiple forms of chicken myelomonocytic growth factor.

The production of chicken myelomonocytic growth factor (cMGF) can be rapidly induced by bacterial lipopolysaccharide from the macrophage cell line HD11. Immunoprecipitation analysis of lipopolysaccharide-induced HD11 cells labeled with various radioactive precursors showed the secretion of a variety of cMGF forms. The precursor-product relationships of the different cMGF forms were studied by pulse-chase experiments, by long-term metabolic labeling in the presence or absence of glycosylation- and oligosaccharide-processing inhibitors, as well as by glycosidase treatment of immunoprecipitates. Our results show that the half-time for intracellular processing/secretion is less than 10 min, making cMGF one of the most rapidly processed proteins. The different forms of the factor are generated from a 24-kDa polypeptide precursor by co- and post-translational acquisition of one or two N-linked oligosaccharides and by O-linked glycosylation. In addition, a fraction of cMGF is modified by long chain, chondroitinase-sensitive, sulfated glycans. This modification is tunicamycin-sensitive, suggesting that the sulfated glycans are attached to N-linked rather than to O-linked oligosaccharides.

Replacement of lys 622 in the ATP binding domain of P100gag-mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v-mil oncogene of MH2 virus.

Lysine 622 in the ATP-binding domain of P100gag-mil, the translation product of the v-mil oncogene of MH2, has been replaced with methionine using oligonucleotide site-directed mutagenesis. This substitution results in the inactivation of the serine/threonine-specific autophosphorylation of P100gag-mil in vitro, indicating that this activity is an intrinsic property of the viral protein. This substitution also suppresses two of the biological properties of MH2 which have previously been shown to be dependant upon the expression of v-mil, namely, the production of chicken myelomonocytic growth factor (cMGF) by v-myc-transformed chicken macrophages and the sustained proliferation of chicken neuroretina cells. These data strongly suggest that the biological properties of v-mil are mediated by the phosphorylation at serine/threonine residues of key cellular substrates. In contrast to the in vitro situation, both the mutant and wild-type proteins appear to be phosphorylated at the same sites and to the same extent in either transformed fibroblasts or macrophages. This, together with the fact that the sites phosphorylated in vivo and in vitro are essentially different indicate that most of the phosphate associated with P100gag-mil in transformed cells does not result from an obligate autophosphorylation event but from the phosphorylation by as yet uncharacterized cellular kinase(s).

Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively.

MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v-myc is required to maintain the transformed phenotype.

V- mil induces autocrine growth and enhanced tumorigenicity in v- myc-transformed avian macrophages

MH2, an avian retrovirus containing the v- myc and v- mil oncogenes, rapidly transforms chick hematopoietic cells in vitro. The transformed cells belong to the macrophage lineage and proliferate in the absence of exogenous growth factors. Here we analyze a series of MH2 deletion mutants and show that these two oncogenes together establish an autocrine growth system in which v- myc stimulates cell proliferation, while v- mil induces the production of chicken myelomonocytic growth factor (cMGF). We also demonstrate that these two oncogenes cooperate in vivo. MH2 efficiently induces monocytic leukemias and liver tumors, while deletion mutants lacking either a functional v- mil or v- myc do not.

Ts mutants of E26 leukemia virus allow transformed myeloblasts, but not erythroblasts or fibroblasts to differentiate at the nonpermissive temperature

The myb,ets-containing avian acute leukemia virus E26 transforms myeloblasts, erythroblasts, and fibroblasts in culture and causes a mixed erythroid/ myeloid leukemia in chicks. We report the isolation and characterization of four E26 mutants that are temperature-sensitive ( ts) for myeloblast transformation. At the permissive temperature, tsE26-transformed myeloid cells resemble macrophage precursors and proliferate rapidly, provided the growth medium contains chicken myelomonocytic growth factor (cMGF). When shifted to the nonpermissive temperature the cells stop growing and differentiate into macrophage-like cells, as determined by their expression of morphological, functional, and antigenic markers of normal macrophages. They also lose their responsiveness to cMGF and secrete a cMGF-like factor. Ts mutants of E26 retain their leukemogenicity and their ability to transform both erythroblasts and fibroblasts at the nonpermissive temperature, suggesting that the myb oncogene of E26 causes myeloblast transformation and that ets is responsible for erythroblast and fibroblast transformation.

Autocrine growth induced by src-related oncogenes in transformed chicken myeloid cells

Chicken myeloid cells transformed by the v-myb- or v-myc-containing leukemia viruses, E26 and OK10, respectively, require chicken myelomonocytic growth factor (cMGF) for proliferation in vitro. Upon superinfection with retroviruses carrying oncogenes of the src gene family, these myeloid cells acquire the ability to grow in the absence of exogenous cMGF. Conditioned medium prepared from superinfected E26 cells contains a growth-stimulating activity similar in biological and immunological properties to cMGF. This activity is reduced by more than 80% following absorption of conditioned media with antiserum against cMGF. Incubation of superinfected E26 cells with an immunoglobulin fraction of antiserum against cMGF inhibits their proliferation, indicating that the cells are dependent on the secreted factor. We conclude that viral oncogenes of the src family can induce chicken myeloid cells to produce a cMGF-like factor(s) that stimulates proliferation of these cells in an autocrine fashion.